Mechanistic insights into Bardet-Biedl syndrome, a model ciliopathy (original) (raw)
An understanding of the molecular defects in BBS has provided insight into the defects of other ciliopathies. At the same time, our expanding understanding of the roles of cilia is both informing the etiopathology of the human phenotypes and unmasking novel, often subtle, clinical defects in patients with BBS and other ciliopathies.
Retinal defects. BBS patients manifest a progressive retinal dystrophy of the photoreceptors, sometimes with early macular involvement (48). Mice in which Bbs genes are modified model this phenotype to a large extent, albeit not at full penetrance. Bbs1-, Bbs2-, Bbs4-, and _Bbs6-_null mice, as well as a knock-in model of a common BBS1 mutation, M390R, display moderate loss of outer nuclear layer (ONL) retinal tissue by 6–8 weeks of age, which correlates, at least in _Bbs2_- and Bbs4-null mice, with increased cell death (49–53). More detailed observations of photoreceptor structure in BBS retinas indicate that, though lamination of the retina is intact, photoreceptor integrity is disrupted in the outer layers (54). This is morphologically similar to the degeneration observed in patients with Alstrom syndrome (55) and LCA (56).
Some reports have suggested that photoreceptor cell death might be due to an underlying defect in the function of the connecting cilium (CC), a structure that links the inner segments (ISs) to the outer segments (OSs) of photoreceptors. In addition to the localization of at least one BBS protein, BBS8, to the CC (23), _Bbs2_-, _Bbs4_-, and _Cep290/Nphp6_-mutant mice show rhodopsin staining in the IS, suggested to reflect defective transport across the CC (49, 57, 58). This is accompanied by an increase in the expression of stress response genes and apoptotic activity in Bbs4 mutants (59).
Some data are consistent with a transport defect, offering at least two mechanistic possibilities. Based on primarily a tissue culture model, it has been demonstrated that some BBS proteins are important for vesicular transport. The complex formed by several BBS proteins, the BBSome, associates with the GDP/GTP exchange factor Rab8, which allows for vesicle trafficking to the base of the cilium (28). Consistent with a possible vesicular model of photoreceptor degeneration, Rab8 mutants display an accumulation of rhodopsin-carrying vesicles at the base of the CC (60). Additionally, localization of Rab8 to the cilium is facilitated by PCM1 (44).
A second possibility may be a defect in IFT across the CC. Mice and zebrafish with mutations in a number of Ift genes, including Ift88, Ift172, Ift52, and Ift57, exhibit a similar loss of photoreceptors (61–64). IFT proteins localize to the basal body in the IS and along the axoneme in the OS (65) and are necessary for IFT transport across the photoreceptor cilium (64); failure of this transport results in cell death (64).
At the same time, some observations are difficult to reconcile with a trafficking model. Rhodopsin and other IS proteins can translocate to the OS through other, CC-independent mechanisms (reviewed in ref. S23). Given the involvement of the cilium in a variety of signaling pathways, it is important to consider other possibilities. For example, BBS4, and probably other BBS proteins, such as BBS11, are involved in proteasomal-mediated degradation (66). Although it is not yet known whether rhodopsin degradation is affected in Bbs mutants, it is plausible that reduced proteasome activity might lead to the accumulation of material in the IS, which can then trigger ER stress and, eventually, apoptosis.
Alternatively, defective Wnt and Sonic hedgehog (Shh) signaling have each been associated with ciliary dysfunction (66–69). It is interesting to note that cyclin D1 is a known target of β-catenin signaling, and one might expect that an attempt of the photoreceptor to reenter the cell cycle will also lead to apoptosis (S24).
Obesity. The penetrance of the obesity phenotype in patients suggests that the BBS proteins play a fundamental role in energy regulation. However, little is known about their actual role(s). _Bbs_-knockout mice and the _Bbs1_-M390R knock-in display obesity phenotypes and increased feeding behavior (49, 50, 52). Additionally, _Bbs4_- and _Bbs6_-null mice exhibit increased blood pressure and increased levels of the circulating hormone leptin (50, 51). The _Bbs1_-M390R mouse is also hyperphagic and hyperleptinemic, with reduced locomotor activity (53). The role of leptin was further linked to the BBS phenotype when a recent study showed not only that Bbs2_-, Bbs4_-, and _Bbs6_-null mice had increased leptin levels but that this may be a result of resistance to exogenous leptin (70).
The role of neuronal and hormonal cues in regulation of feeding was further explored in _Bbs2_- and _Bbs4_-null mice where the neuronal ciliary localization of the G protein–coupled receptor melanin-concentrating hormone receptor 1 (MCHR1), which regulates feeding behavior, was perturbed (71). Consistent with this finding, genetic screens of the C. elegans mutant tub1, an ortholog of the Tubby gene, indicate that alleles of a fat storage gene, kat1, may be involved in the obesity phenotype, and screens of kat1 mutants indicate that bbs1 may be perturbed in ciliated neurons that sense nutrient levels (72).The role of BBS proteins in the regulation of feeding may be related to IFT, as animals with knockout of either Ift88 or Kif3a were obese, with increased insulin and leptin levels, and these phenotypes may be specific to defects in hypothalamic neurons responsible for regulation of feeding (73, 74). Loss of cilia specifically in proopiomelanocortin (POMC) neurons by POMC:Cre deletion results in an increase in weight and adiposity, though these increases were not as severe as those seen as a result of systemic ablation (73, 74). These findings support a role for cilia in hypothalamic neurons in the brain’s mediation of feeding behavior by interpretation of signals from various organs transmitted to the CNS, including hormonal satiety cues such as leptin and insulin. Leptin excites POMC neurons in the presence of high glucose levels to signal reduced food intake (75). If POMC neurons malfunction, however, the detection of leptin and the subsequent reduction in intake could be defective.
Another mechanism possibly contributing to the observed obesity phenotype involves adipogenesis. Bbs1–4, Bbs6–8, Bbs9, and Bbs11 are all expressed during mouse adipogenesis (76), suggesting they may play a role in the generation of fat tissue. Other ciliary proteins have also been implicated in this process, including retinitis-pigmentosa GTPase regulator interacting protein 1–like (RPGRIP1L), which localizes to the basal body (77) and whose expression is decreased in the adipose tissue of mutants for the adjacent FTO gene (78) and the Alström syndrome gene ALMS1, for which obese knockout mice have been generated (79, 80). The ALMS1 protein, which localizes to the basal body (81, 82), is expressed in the early phases of adipogenesis and may be involved in the conversion of preadipocytes to adipocytes (83). It remains to be determined whether hypothalamic dysfunction alone is sufficient to induce obesity in BBS and other ciliopathies. The attenuated phenotype of the POMC:Cre Ift88 and Kif3a mutants can be explained either by the fact that some leptinergic neurons might have escaped inactivation or that there is a systemic contribution. In addition, it will be important to measure energy expenditure in ciliary mouse mutants and human patients, since a contribution of defective sensing of energy expenditure cannot yet be excluded. Ultimately, systematic tissue–specific ablation and crossing of the mutant animals will be required to answer this question comprehensively.
Polydactyly. Polydactyly in BBS and other ciliopathies is intriguing because of the known mechanisms of digit formation and the involvement of SHH signaling. Digit formation starts in the zone of polarizing activity (ZPA), a structure in the posterior mesenchyme of the limb (or fin) bud that is common in vertebrates (S25). SHH is found in the vertebrate limb bud, including the zebrafish fin bud, and regulates the ZPA (S26, S27). The targets of SHH signaling are the glioma (GLI) transcription factors GLI1, GLI2, and GLI3 (84). When SHH binds to the Patched1 (PTCH1) receptor, Smoothened (SMO) is derepressed, blocking processing of GLI3 from its activator to repressor forms (S28). In the context of limb formation, SHH regulates digit number and identity (S29), and either ectopic SHH expression or loss of GLI3 causes polydactyly (S30–S32).
Several lines of evidence implicate the cilium in SHH signaling and digit formation, not least of which is the localization of both SMO and PTCH1 to the cilium, where PTCH1 inhibits SMO by preventing its accumulation at the cilium (68, 69). SHH binding of PTCH1 causes it to be mislocalized from the cilium so that SMO can be activated there (69) (Figure 2). IFT protein function is required for SHH signaling. Mouse mutants for Ift172 and Ift88 exhibit phenotypes consistent with defects in SHH signaling, including loss of ventral neural cell populations and preaxial polydactyly (85). The defect appears to be downstream of PTCH1 and SMO, possibly at the level of GLI processing. A later study confirmed that the defect lies in the proteolytic processing of GLI3 to its repressor form, which requires IFT172 (86). Another study of Ift88 mutants revealed that GLI2 and GLI3 — in addition to a negative regulator of SHH, Sufu — also localize to the cilium in the developing limb bud and require IFT88 to do so (87).
Signaling pathways in the cilium. (A) Hedgehog signaling is regulated by the Ptch1 and Smo receptors. Binding of the hedgehog ligand to Ptch1 alleviates its inhibition of transport of Smo into the cilium, where it may regulate IFT involvement in Gli processing to either its activator form (GliA) or repressor form (GliR). Downstream targets of Gli-directed transcription include limb/digit formation, neurogenesis, and neural development. (B) Canonical and noncanonical Wnt signaling components are associated with the cilium and basal body. Binding of a canonical Wnt ligand to Frizzled (Fz) and LPL-related protein (Lrp) receptors causes Dishevelled-mediated (Dvl-mediated) recruitment of the β-catenin destruction complex to the plasma membrane, preventing β-catenin degradation. β-Catenin can then regulate T cell factor– and lymphoid-enhanced binding factor–mediated (TCF- and LEF-mediated) transcription of targets in the nucleus and subsequent processes such as cell proliferation and specification of cell fates. In the absence of ligand, Dvl and the β-catenin destruction complex exist freely in the cytoplasm and can target β-catenin for proteasome-mediated degradation. BBS proteins regulate proteasome function in addition to the transport of Inversin (Inv) from the basal body into the cytoplasm, where it can reduce cytoplasmic levels of Dvl via phosphorylation, a process that can also be regulated by Kif3a. Binding of a noncanonical Wnt ligand also recruits Dvl to the Fz receptor, a process regulated by Inv. This initiates activation of downstream targets, including the PCP effectors RhoA and JNK, and downstream processes that affect the actin cytoskeleton, cell adhesion, and cell polarity. Regulation of PCP signaling also occurs at the level of Dvl and its interactions with Vangl2 in the cilium and inturned (Int) and fuzzy (Fy) at the basal body.
Additional findings support the role of IFT in Shh signaling: IFT proteins regulate both activator and repressor Gli expression (88), and suppression of retrograde IFT results in mislocalization of SMO from the cilium and disruption of Gli3 processing (89). It also appears that the interaction of SMO and the IFT protein kinesin family member 3A (KIF3A) is regulated by β-arrestin (90). Loss of KIF3A in cartilage results in skeletogenic defects (91), consistent with the role of IFT in regulating limb formation, possibly through SHH. Taken together, these findings provide strong evidence that IFT may regulate Shh signaling in limb bud cells and that defects in this regulation results in aberrant formation of digits.
BBS proteins have been linked to Shh regulation of limb development as well. Expression of bbs7 is enriched in the developing zebrafish fin bud (32). Furthermore, exogenous misexpression of either bbs1 or bbs7 results in increased Shh expression in the anterior ZPA and skeletal pattern changes in the pectoral fin consistent with a link between BBS proteins and Shh-directed limb development (92). It is notable, however, that the polydactyly associated with BBS is almost always postaxial. While other disorders associated with postaxial polydactyly, notably Pallister-Hall syndrome (S33), arise from defects in Shh signaling, the possibility exists that at least one other signaling pathway is involved, the Wnt pathway. This is because mice lacking Dkk1, an extracellular protein that binds the LRP5/6 receptor to antagonize canonical Wnt signaling, also exhibit postaxial polydactyly, as do hypomorphic Dkk1 mutants (93, 94). Loss of BBS protein function results in defects in suppression of noncanonical Wnt signaling, with a slight increase in targets of canonical Wnt signaling (66). Thus, the suppression of BBS proteins may have the net result of producing defects similar to those seen in mice with upregulation of canonical Wnt signaling.
Renal dysfunction. The low incidence of renal function abnormalities in BBS patients and mouse models, relative to other features of the disorder, has made this phenotype one of the less well-investigated aspects of disease. However, other ciliopathies exhibit a range of renal phenotypes (Figure 3), which, in addition to reports of kidney defects in ciliary mouse mutants, provides some insight into the mechanisms underlying the renal phenotype. Notably, the hypomorphic Ift88 mouse mutant orpk provided a model for PKD as a result of defective cilia assembly (95). Other ciliary proteins have been associated with kidney disease as well (reviewed in ref. S34).
Renal phenotypes of ciliopathies. (A) Intravenous pyelogram of BBS kidney showing pyelocalyceal dilatation, calyceal clubbing, and pericalyceal cysts. (B) ADPKD kidneys typically have numerous cysts of variable sizes, with even distribution throughout the renal cortex and medulla. (C) ARPKD kidneys retain a reniform configuration with radiating, fusiform nonobstructive dilatations of the collecting ducts extending from the medulla to the cortex. (D) In NPH — as in JBTS, Senior-Loken Syndrome, and Jeune syndrome — the kidney exhibits cysts arising from the corticomedullary junction. (E) MKS kidneys manifest cystic dysplastic changes. Images reproduced with permission from Radiology (125) (A), Journal of the American Society of Nephrology (113) (D), and Advances in Anatomic Pathology (126) (B, C, and E). Scale bar: 1 cm.
Several lines of evidence support a role for BBS proteins, including the direct interaction of BBS1, -2, -4, and -7 with proteins present in the kidney (96). _Bbs4_-null mice display shorter renal tubule cilia initially, and these become longer over time, indicating a defect of either cilia assembly (97) or maintenance/regulation of ciliary length. Compelling evidence implicating BBS proteins in kidney phenotypes was exhibited with the formation of kidney cysts in bbs zebrafish morphants (98). Interestingly, the cyst phenotype could be rescued by culturing embryos in a solution containing the mTOR signaling inhibitor rapamycin (98). These findings provide support for a possible role for pathways upstream of mTOR in the kidney phenotype, most notably the noncanonical Wnt planar cell polarity (PCP) pathway.
BBS proteins, as well as other ciliary proteins, have been implicated in PCP signaling. Suppression of BBS protein function in mice or zebrafish, for example, produces defects reminiscent of the classical phenotypes resulting from mutations in PCP genes (including Vangl2), such as neural tube defects, open eyelids, perturbation of cochlear stereociliary bundles, and disruption of convergent extension movements (99). Furthermore, BBS genes interact with core PCP mutants, underscoring their role, and the role of cilia, as key regulators of the pathway. Further investigation revealed the interaction of BBS proteins with noncanonical Wnt ligands (Wnt5 and Wnt11), which, when perturbed, produces convergent extension defects and stabilization of cytoplasmic levels of β-catenin as a result of defective proteasome function (66). This defect may be linked to IFT function, as it is phenocopied by suppression of kif3a (66), which suppresses canonical Wnt signaling by blocking the casein kinase–mediated phosphorylation of the scaffold protein dishevelled (67). Interestingly, disruption of PCP produces defects in ciliogenesis as well. Perturbation of the PCP effectors inturned and fuzzy results in defects in ciliogenesis and convergent extension as a result of the interaction with dishevelled at the basal body (100). These findings are consistent with a role of cilia in regulation of PCP signaling and vice versa (Figure 2).
This is particularly relevant to the renal disease phenotype because the PCP pathway regulates cell polarity and orientation during the development of the nephron; cell mitotic spindles must be oriented properly during proliferation and tubule extension (101). Cilia have been implicated in this process, not least because loss of cilia in kidney, as induced by Kif3a knockout specific to renal tubular epithelial cells via Ksp:Cre, demonstrated that loss of PCP-dependent mitotic spindle orientation produces cysts as a result of proliferation defects (102). Similarly, ablation of Ift20 in collecting duct cells produces cystic kidneys as a result of failure of proliferating cells to properly orient their mitotic spindles along the tubule (103). Another model of PCP defects, the _Fat4_-null mouse, also forms cystic kidneys as a result of aberrant mitotic spindle orientation (104). These findings are consistent with the finding that loss of Pkhd1, a basal body protein associated with autosomal recessive PKD (ARPKD) (105), disrupts PCP signaling (101).
In addition to the association of PCP defects with renal pathology, β-catenin–dependent canonical Wnt signals are widely believed to be important for kidney development. Wnt9b and Wnt4 have been implicated in the specification of epithelial cells to a renal fate (106, 107), and treatment of isolated rat kidney mesenchyme with the GSK3-β inactivators lithium or 6-bromoindirubin-3′-oxime induces nephron differentiation (108), indicating that excessive β-catenin promotes the process. This has also been demonstrated in vivo in mice, where conditional loss of β-catenin in kidney targeted to ureteric cells results in defects in branching morphogenesis and renal dysplasia (109).
There is evidence that regulation of the canonical Wnt pathway is linked to the cilium, especially with respect to kidney phenotypes. For example, cells of the developing mouse kidney lacking cilia as a result of conditional Ift20 knockout, which leads to cystic kidneys, exhibit increased intracellular canonical Wnt signaling (103). Consistent with this, in the zebrafish, loss of seahorse (human homolog: LRCC6) results in cystic kidneys as a result of its interaction with the ciliary gene inversin and regulation of dishevelled (110). This is important in the context of Wnt signaling because inversin seems to function as a switch between canonical and noncanonical Wnt regulation in the kidney (111) (Figure 2). It is possible, then, that cilia regulate the dissemination of Wnt signaling in the cell, which directs downstream targets to drive cell fate specification and morphogenesis in the developing kidney.
The role of cilia in Wnt signaling suggests that defects in this pathway underlie the overlapping kidney phenotypes across the ciliopathies. However, there are distinct differences in both quality and severity of defects across ciliopathies (Figure 3). For example, reports of kidney phenotypes in BBS patients have included hyperplasia, dysplasia, and cystic kidneys (112). Other disorders, however, exhibit only hyperplasia and severely cystic kidneys (ARPKD) or progressive dysplasia with localized cysts (NPH) (33, 113). Furthermore, some ciliopathies, such as AS and NPH, manifest other phenotypes, such as fibrosis. The varying severity and presentation of additional phenotypes suggest that proteins underlying these disorders may either have additional cellular functions or participate in different components of the same pathways. Regarding the first possibility, one example lies in the fact that upregulation of growth factors, such as EGF and TGF, is thought to contribute to the hyperplasia and cysts associated with ARPKD (114); the involvement of TGF also suggests a link to the fibrosis phenotype (115). If either hypothesis proves to be true, this would implicate ciliary proteins in regulation of growth factor pathways at the cellular level, though a direct link has yet to be demonstrated. Finally, with respect to the differential severity of perturbation of intracellular pathways by perturbation of ciliary proteins, the link between BBS and NPH may shed light onto the varying severity produced by targeting a signaling pathway differentially. One protein underlying NPH is inversin (NPHP2), which targets dishevelled for degradation to prevent β-catenin–dependent proliferation (111). Mutations in inversin may partially affect its function; however, mutations in BBS disrupt retrograde IFT and, subsequently, may disrupt transport of inversin to the cytoplasm altogether, completely preventing its interaction with dishevelled (112). Thus, the specific step disrupted in a pathway could dictate the severity of the resulting cellular, and physiologic, defect.
Mental retardation. One of the least-understood ciliopathy phenotypes is mental retardation. Behavioral abnormalities have been reported in _Bbs_-knockout mice, but the underlying physical defect has not been explored in detail. Though the exact function of ciliary proteins in brain development is unclear, expression of at least one BBS gene, Bbs3/Arl6, which is involved in ciliary transport (25, 116), is seen in developing neural tissues (117). Furthermore, consistent with cerebral anomalies observed in BBS patients (118), the _Bbs1_-M390R knock-in mouse had several morphological defects in the brain, including ventriculomegaly of the lateral and third ventricles, a thin cerebral cortex, and reduced corpus striatum hippocampus (53). Additionally, cilia along the enlarged third ventricle, though intact, were elongated and swollen at the distal end, suggestive of IFT defects.
There is evidence linking cilia to two processes in neural development: neurogenesis and neuronal migration. Recent evidence has linked Shh signaling, regulated by the cilium, to neurogenesis and hippocampal development. Conditional ablation of Kif3a in hGFAP:Cre cells results in defects in postnatal neurogenesis in the dentate gyrus, and this process is apparently regulated by Smo at the cilium, as conditional hGFAP:Cre;Smo mutants show similar defects, and constitutively active Smo was not able to rescue the conditional Kif3a phenotype (119). Furthermore, loss of cilia due to specific ablation of the ciliary gene Stumpy in hippocampal astrocyte-like neural precursors results in gross morphological abnormalities as a result of defects in neuronal precursor proliferation and defective Shh signaling (120). The involvement of Shh signaling at the cilium in proliferation of developing neuronal populations provides strong evidence that defective cilia play an important role in properly populating the hippocampus.
In addition to the generation of neurons during development, the migration of those neurons to various regions is also affected in BBS and potentially other ciliary mutants. BBS modulation of Wnt signaling may play a role in regulating movement of neuronal precursors. Loss of the zebrafish PCP protein strabismus (stbm), in Trilobite mutants results in defects in cell polarity and resulting cell movements, including migration of hindbrain neurons (121). Similar migration defects result in suppression of another PCP protein, Prickle1, which interacts with stbm (122). Similarly, BBS proteins interact with stbm in Trilobite embryos, suggesting that this interaction plays a role in BBS neuronal phenotypes (99). The involvement of BBS proteins and the cilium in regulation of PCP signaling suggests that cilia may play a role in cell movements during development and, specifically, the migration of neurons during brain development.